The activity of Rho family GTPases is regulated by over 80 GTPase exchange factors (GEFs). Each GTPase is activated by multiple GEFs, each GEF can interact with several different Rho GTPases, and different interactions occur at specific times and places within cells. Hence, deciphering the GTPase signaling programs that control particular cell functions has been a formidable challenge. We conjecture that this can be addressed effectively only by directly observing the coordinated activities of Rho GTPases and their associated regulators in living cells. Building on the biosensor technology developed in Project 1, in this project we will establish techniques to dissect the relationships between multiple GEFs and Rho GTPases; i) by developing the ability to simultaneously observe multiple GEF and Rho GTPase activities in the same living cells; ii) by inhibiting or activating specific network proteins with light while observing others and iii) by computationally integrating the data from multiple experiments that encompass different GEF-GTPase combinations, to generate a quantitative model of the activation hierarchy and kinetics of these signaling networks. As a driving biological problem we will focus on modeling Rho GTPase signaling programs that regulate actin cytoskeleton dynamics and adhesion formation at the protruding edge of cells undergoing directed migration. The same techniques will enable Projects 3 and 4 to investigate signaling programs in the context of collective cell migration and mechanotransduction. The specific aims for the collaborative work between the Danuser and Hahn labs in this project are: 1) Imaging the activity of GTPases and associated GEF(s) in the same living cell. 2) Implementing time series analysis methods to integrate the data from multiple experiments, each using different combinations of GEF - GTPase pairs, into a consistent model of GEF - GTPase interaction networks. 3) Establishing GEF-GTPase interaction networks in Rac1/Cdc42 signaling programs dunng cell protrusion and retraction events.